The CARBA-MAP study: national mapping of carbapenemases in Spain (2014–2018)

Introduction Infections caused by carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa, including isolates producing acquired carbapenemases, constitute a prevalent health problem worldwide. The primary objective of this study was to determine the distribution of the different carbapenemases among carbapenemase-producing Enterobacterales (CPE, specifically Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and Klebsiella aerogenes) and carbapenemase-producing P. aeruginosa (CPPA) in Spain from January 2014 to December 2018. Methods A national, retrospective, cross-sectional multicenter study was performed. The study included the first isolate per patient and year obtained from clinical samples and obtained for diagnosis of infection in hospitalized patients. A structured questionnaire was completed by the participating centers using the REDCap platform, and results were analyzed using IBM SPSS Statistics 29.0.0. Results A total of 2,704 carbapenemase-producing microorganisms were included, for which the type of carbapenemase was determined in 2692 cases: 2280 CPE (84.7%) and 412 CPPA (15.3%), most often using molecular methods and immunochromatographic assays. Globally, the most frequent types of carbapenemase in Enterobacterales and P. aeruginosa were OXA-48-like, alone or in combination with other enzymes (1,523 cases, 66.8%) and VIM (365 cases, 88.6%), respectively. Among Enterobacterales, carbapenemase-producing K. pneumoniae was reported in 1821 cases (79.9%), followed by E. cloacae complex in 334 cases (14.6%). In Enterobacterales, KPC is mainly present in the South and South-East regions of Spain and OXA-48-like in the rest of the country. Regarding P. aeruginosa, VIM is widely distributed all over the country. Globally, an increasing percentage of OXA-48-like enzymes was observed from 2014 to 2017. KPC enzymes were more frequent in 2017–2018 compared to 2014–2016. Discussion Data from this study help to understand the situation and evolution of the main species of CPE and CPPA in Spain, with practical implications for control and optimal treatment of infections caused by these multi-drug resistant organisms.

Introduction: Infections caused by carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa, including isolates producing acquired carbapenemases, constitute a prevalent health problem worldwide.The primary objective of this study was to determine the distribution of the different carbapenemases among carbapenemase-producing Enterobacterales (CPE, specifically Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and Klebsiella aerogenes) and carbapenemase-producing P. aeruginosa (CPPA) in Spain from January 2014 to December 2018.
Methods: A national, retrospective, cross-sectional multicenter study was performed.The study included the first isolate per patient and year obtained from clinical samples and obtained for diagnosis of infection in hospitalized patients.A structured questionnaire was completed by the participating centers using the REDCap platform, and results were analyzed using IBM SPSS Statistics 29.0.0.
Results: A total of 2,704 carbapenemase-producing microorganisms were included, for which the type of carbapenemase was determined in 2692 cases: 2280 CPE (84.7%) and 412 CPPA (15.3%), most often using molecular methods and immunochromatographic assays.Globally, the most frequent types of carbapenemase in Enterobacterales and P. aeruginosa were OXA-48-like, alone or in combination with other enzymes (1,523 cases, 66.8%) and VIM (365 cases, 88.6%), respectively.Among Enterobacterales, carbapenemase-producing K. pneumoniae was reported in 1821 cases (79.9%), followed by E. cloacae complex in 334 cases (14.6%).In Enterobacterales, KPC is mainly present in the South and South-East regions of Spain and OXA-48-like in the rest of the country.Regarding P. aeruginosa, VIM is widely distributed all over the country.Globally, an increasing percentage of OXA-48-like enzymes was observed from 2014 to 2017.KPC enzymes were more frequent in 2017-2018 compared to 2014-2016.

Introduction
Multiple antibiotic resistance has emerged as major public health threat.Infections caused by antibiotic-resistant bacteria are associated with significant morbidity and mortality (Prestinaci et al., 2015;Li et al., 2022).Currently the major problem is caused by multi-drug resistant (MDR) Gram-negative bacteria, particularly Enterobacterales, Pseudomonas aeruginosa or Acinetobacter baumannii presenting resistance to carbapenems (Sheu et al., 2019;Jean et al., 2022;Tenover et al., 2022).All these microorganisms are included in the WHO list of priority pathogens that urgently require investigation and development of new and effective antibiotic 10.3389/fmicb.2023.1247804Frontiers in Microbiology 03 frontiersin.orgtreatments (Tacconelli et al., 2018).For years, multidrug resistance in Enterobacterales has been related to the production of extendedspectrum β-lactamases (ESBLs), for which carbapenems have been considered first-line therapeutic options.However, their use has led to a more serious problem: the emergence of carbapenem-resistant Enterobacterales (CRE; Paño Pardo et al., 2014;van Duin and Doi, 2017;Suay-Garcia and Perez-Gracia, 2019).Similarly, there has been an increasing prevalence of infections produced by MDR and extensively drug-resistant (XDR) P. aeruginosa, which results from the extraordinary ability of this organism to develop resistance to nearly all available antibiotics through the selection of mutations in chromosomal genes or the acquisition of mobile genes (Del Barrio-Tofiño et al., 2019;Horcajada et al., 2019).
The primary objective of this study was to determine the prevalence of carbapenemase enzymes among CPE and CPPA in Spain over a five-year period (January 2014 to December 2018).The secondary objectives were to describe the demographic characteristics of the infected patients, to determine the different types and subtypes of carbapenemases and their geographic distribution in Spain, and to illustrate the evolution of carbapenemases throughout the study period.

Materials and methods
Data from CPE and CPPA were collected in a National retrospective multicenter (30 Spanish hospitals) study, from January 2014 to December 2018.
The first isolate of carbapenemase-producing E. coli, K. pneumoniae, E. cloacae complex, K. (E.) aerogenes, or P. aeruginosa per patient and year cultured from clinical samples obtained for diagnosis of infection in hospitalized patients was included.Isolates obtained from surveillance samples were excluded.Bacterial identification and susceptibility testing had been performed in the participating centres using combined identification and antibiogram panels by automated commercial systems (Table 1).Carbapenemase detection and identification of the major families were determined using established phenotypic and genotypic methods (see below) in the participating centres, as shown in Table 2. Briefly, mCIM (modified 10.3389/fmicb.2023.1247804Frontiers in Microbiology 04 frontiersin.orgcarbapenem inactivation method), Hodge Test and/or Carba-NP test were used as screening methods for carbapenemase detection, and immunochromatography and/or molecular methods were the main methods for carbapenemase type identification.Information about the particular allele of carbapenemase determined by molecular methods (in house PCR or sequencing) was reported in few cases (Table 3).
A structured questionnaire was completed (details presented in Supplementary Table S1), which included general questions (study site identification, susceptibility testing methodology, use of automated antibiogram methods, susceptibility criteria (EUCAST or CLSI) for definition of clinical categories, methods for carbapenemase detection) and questions related to the included patients/microorganisms (demographic data, sample type, service of hospitalization, bacterial species, type of carbapenemase and, when available, specific carbapenemase allele) All data were registered by researchers using the REDCap platform.
The results were analyzed using IBM SPSS Statistics 29.0.0.Age is expressed as average, median and range.The results are expressed as number of cases (n) and percentage (%) in all the variables.
The study was classified by the Spanish Agency of Drugs and Pharmaceutical Products (AEMPS as in Spanish) as a "Post authorization Study with Other designs different to the Prospective follow-up" (EPA-OD, as Spanish translation) with protocol code MSD-CAR-2020-01.Based on the classification of Post authorization Study, and following the Spanish legislation, "Orden SAS 2470/2009." The study was reviewed and approved by the Hospital Universitario Reina Sofía Ethics Committee on 29 July 2020.
Different phenotypic and genotypic methods were used in the participating centres for detecting and identifying carbapenemase types, as shown in Table 2. Phenotypic screening assays (including mCIM test, modified Hodge test or colorimetric methods) were used in 500 (18.5%)cases.As previously indicated, in 12 (0,4%) cases only a screening method was performed for carbapenemase detection.This was the case for 1 E. coli, 8 K. pneumoniae, 1 K. aerogenes, and 2 P. aeruginosa.The most frequent techniques used for identifying the major families of carbapenemases were molecular methods, in 1.712 (63.6%) cases, followed by immunochromatography in 887 (32.9%) cases, alone or in combination with different methods as synergyinhibitors.Between 2014-2017, molecular techniques were the most frequent methods used (63.7-73.2%),but in 2018 immunochromatography methods were used more frequently compared to molecular methods (53.3 vs. 47.4%).
The distribution of carbapenemase types by bacterial species is shown in Table 3. Globally, the most frequent types of carbapenemases in Enterobacterales were OXA-48-like, either alone or in combination with other enzymes (1.523 cases, 66.8%) and VIM (365 cases, 88.6%) in P. aeruginosa.
The geographical distribution of carbapenemase types is presented in Figures 1A,B.In Enterobacterales, KPC is mainly present in the South and Southeast of Spain, while OXA-48-like is prevalent in the rest of the country.Regarding P. aeruginosa, VIM is widely distributed throughout the country.
The temporal distribution of carbapenemase types is presented in Figure 2.An increasing number of organisms producing OXA-48-like enzymes was observed from 2014 to 2017.Organisms producing KPC enzymes were more frequent in 2017-2018 compared to 2014-2016.
Table 1 shows the distribution of methods used by the participating centres for antimicrobial susceptibility testing.In most cases (2.501, 92.5%), antimicrobial susceptibility testing of the corresponding organism was performed using commercial broth microdilution panels.Diffusion methods were the only susceptibility testing assays performed in 196 (7.2%) cases.The MicroScan and Vitek systems were used in 1.649 cases (61%) and 709 cases (26.2%), respectively.Either disk-diffusion or gradientstrips diffusion assays were performed in 146 (5.4%) and 274 (10.1%) cases, respectively, and both assays were performed simultaneously in an additional 98 (3.6%) cases, additionally to commercial broth microdilution panels.
Clinical categories were most often defined following EUCAST recommendations (78.9% of cases), considering the criteria established at the time when the corresponding organism was evaluated.Clinical categories of tested antimicrobial agents against the different microorganisms are presented in Table 4.For Enterobacterales, more than 90% of all isolates were resistant to piperacillin-tazobactam, cefotaxime and ceftazidime.Resistance to ertapenem (85.4%) was higher than to meropenem and imipenem (47.3 and 41.5%, respectively).In the case of P. aeruginosa, the highest percentages of resistance (excluding cefotaxime and ertapenem) were observed for cefepime (90.8%), ceftazidime (90.5%), meropenem (89.7%), ciprofloxacin (86.3%) and piperacillin-tazobactam (85.1%) and imipenem (81.0%).
The percentages of resistance to different antimicrobials by type of carbapenemase are shown in Table 5.For Enterobacterales, resistance rates to carbapenems varied considerably depending on the specific type of carbapenemases, but it was in practically all cases higher for ertapenem.The lowest percentages of resistance of the tested compounds were noted for the non-β-lactam colistin and amikacin, both in Enterobacterales and in P. aeruginosa.Resistance rates to imipenem and meropenem were particularly low for GES-producing isolates.Interestingly, resistance to aztreonam in isolates producing NDM was as high as 93.9%, with values of 49.2% for VIM producers and 33.3% for IMP producers.

Discussion
This study represents an effort to evaluate the frequency of the different carbapenemase enzymes among CPE (including not only K. pneumoniae and E. coli, but also K. aerogenes and E. cloacae complex) and CPPA over an extended period and in a large number of hospitals in Spain.Previous studies have focused on organisms isolated during a brief period or concentrated on a limited number of species (Miro et al., 2013;Oteo et al., 2015;Del Barrio-Tofiño et al., 2017;Grundmann et al., 2017;Del Barrio-Tofiño et al., 2019;Horcajada et al., 2019;Cañada-García et al., 2022).Most Spanish laboratories have implemented methodologies for carbapenemase identification, at least to family/type level.The most frequently used methods were commercial PCR or in-house PCR assays between 2014 and 2017 (63.7-73.2%).In many cases, these were supplemented with immunochromatographic detection of major carbapenemase families and phenotypic/colorimetric tests.Different multiplex PCR commercial system were used during the study period including genes of the most frequent carbapenemases (e.g., KPC, VIM, IMP, NDM…).These systems are time-saving, have high levels of sensitivity and specificity but are quite expensive and miss to identify less frequent carbapenemases (e.g., GES, IMI…) or specific alleles (e.g., IMP-19 or IMP-66 in GenXpert system; Cui et al., 2019;Kanahashi et al., 2021).It should be noted that in the present study the use of in-house PCR and whole genome sequencing methodology has increased during 2018 (7%) compared to the previous years.Actually, this methodology is useful to report information about specific alleles and epidemiological information.However, it is not available in all microbiology laboratories, which explains the increased use of methods based on immunochromatography.In the present study, despite the fact that molecular methods have been used more frequently during the 5 years, the use of immunochromatography has increased over the years of the study (18.6-53.3%)because of price, reliability and easy-to-perform method, as opposed to molecular methods.These approaches are essentials not only for a more effective control of carbapenemase-producing organisms but also for guiding appropriate therapy of patients infected with these bacteria, especially considering the availability of new agents (particularly new β-lactam/ β-lactamase inhibitor combinations) which have significant differences in their spectrum of activity against CPE and CPPA.MIC values of clinically relevant antimicrobial agents are usually determined in our country using commercial semi-automatic methods, particularly MicroScan WalkAway (Beckman Coulter) and to a lesser extent, Vitek (bioMérieux; Table 1).A minority of laboratories used only the disk-diffusion assay, either alone or combined with gradient strips, when testing CPE and CPPA.The clinical categories of the tested compounds are largely defined using EUCAST criteria, which is likely influenced by the multiple activities and reports from the Spanish national antimicrobial susceptibility testing committee (CoEsAnt, website: http://coesant-seimc.org; Cantón et al., 2020;Larrosa et al., 2020Larrosa et al., , 2022)).
The results from this study indicate that the most frequent carbapenemases in Enterobacterales in Spain over the study period are OXA-48-like and KPC, which is consistent with previous studies conducted in recent years (Miro et al., 2013;Oteo et al., 2015;Grundmann et al., 2017;Cañada-García et al., 2022).The most common species among CPE have been K. pneumoniae and E. cloacae complex, with OXA-48-like enzymes being the most frequent carbapenemases in K. pneumoniae (also observed in E. coli).In E. cloacae complex, VIM-type enzymes have been the most frequent.VIM enzymes have also been the most common carbapenemases in P. aeruginosa, which is consistent with the results of previous studies in Spain (Del Barrio-Tofiño et al., 2017, 2019;Horcajada et al., 2019).
It is important to note that this study has focused on carbapenemase-producing organisms rather than carbapenemresistant gram-negative bacteria.Recent reports from our country (Miro et al., 2013;Oteo et al., 2015;Del Barrio-Tofiño et al., 2017, 2019;Grundmann et al., 2017;Horcajada et al., 2019;Cañada-García et al., 2022) have already demonstrated that while carbapenem resistance in K. pneumoniae and E. coli is mainly related to carbapenemase production, carbapenem resistance in P. aeruginosa and E. cloacae complex is usually related to the production of β-lactamases with a low/moderate hydrolytic efficiency of carbapenems, combined with reduced intrabacterial drug accumulation due to porin loss and/or overexpression of efflux pumps (Del Barrio-Tofiño et al., 2017, 2019;Horcajada et al., 2019).
In addition to OXA-48-like enzymes in Enterobacterales and VIM carbapenemases in E. cloacae complex and P. aeruginosa, KPC has also been quite common among K. pneumoniae isolates, while NDM, IMP and GES have been found less frequently.The recently published CARB-ES-19 study (Cañada-García et al., 2022) has documented that the main KPC allele disseminated in Spain is KPC-3, but other studies have also identified new KPC (Hernández-García et al., 2021, 2022;Guzmán-Puche et al., 2022) responsible for resistance to ceftazidimeavibactam which, in some cases, cause collateral susceptibility to carbapenems.
From 2014 to 2017, there has been a continuous increase in the identification of isolates (mostly K. pneumoniae) producing OXA-48like enzymes, although the number of isolates with this type of enzymes stabilized in 2018.In contrast, the number of KPC-producing organisms (in all cases Enterobacterales, as this enzyme was not identified in P. aeruginosa in our study), increased in 2017 and particularly in 2018 when it became the second most frequent β-lactamase in Spain, as also observed in the CARB-ES-19 study which included carbapenemase-producing K. pneumoniae and E. coli (Cañada-García et al., 2022).An analysis of the geographical distribution of the most frequent carbapenemases in Spain indicates that in Enterobacterales, KPC seems to be more prevalent in the Southern and South-eastern regions of the country, while OXA-48-like is more prevalent in the remaining regions.These data contrast with the findings of a 2015 report (with organisms collected between February and May 2013;Oteo et al., 2015) which indicated that many provinces were free from OXA-48like producing Enterobacterales, and KPC producers were identified in just two central provinces and in one Eastern province.For P. aeruginosa, VIM is widely distributed all over the country.
In the CPE cases, OXA-48-like strains presented high resistance level to piperacillin-tazobactam and cephalosporins (cefotaxime, ceftazidime and cefepime) suggesting those strains are associated to ESBL production.Interestingly, resistance to aztreonam in CPE was between 33 and 50% except for NDM which was over 93%.On the other hand, in CPPA resistance to aztreonam was under 32% but in IMP and other metallo-β-lactamases was under 12%.Therefore, identification of metallo-carbapenemase type prior to antimicrobial susceptibility testing information is available can be useful for empiric treatment in a high proportion of cases.Resistance to colistin was under 17% in all CPE and under 5.5% in all types of metallo-βlactamases in CPPA thus being a therapeutic option.GES producers showed quite low levels of resistance to imipenem or meropenem according to their low hydrolytic activity, but >50% resistance to ertapenem so it should be included in susceptibility testing methods.
This study has several limitations, most of which are inherent to its retrospective design.While we have identified the carbapenemase families of major epidemiological and clinical relevance, in most cases the specific alleles have not been defined, as sequencing data were not available from the participating centers.MICs of carbapenems and other agents were determined in most centres using commercial panels, which, in many cases, do not include the necessary number of dilutions to allow precise MIC definition or the correct application of EUCAST clinical breakpoints.We suggest that manufacturers follow the CoEsAnt recommendations (Zhiyong et al., 2021) for the ideal selection of agents to be used in panels for automated systems.The use of commercial panels also resulted in a lack of information on new agents with activity against carbapenemase producing organisms (such as ceftazidimeavibactam, meropenem-vaborbactam, imipenem-relebactam, cefiderocol, eravacycline, etc.) since these panels did not include the indicated drugs.A new study considering these new therapeutic options is warranted.Finally, only the most frequent CPE species have been considered, and no information on organisms of genera Citrobacter, Proteus, Providencia, Morganella, Hafnia, etc. was obtained.Similarly, our study only considered P. aeruginosa, although some reports also indicate the importance of other species, particularly P. putida and related species, as carbapenemase producers (Gilarranz et al., 2013;Molina et al., 2014;Ocampo-Sosa et al., 2015).There is also a limitation in detecting less prevalent carbapenemases as GES enzymes by immunochromatographic or PCR assays.In Spain, GES enzymes have been described in P. aeruginosa in some reports (Herrera-Espejo et al., 2022;Recio et al., 2022), but in this study maybe less represented because of assays limitations.
The information from this study help to understand the situation and evolution of the main species of carbapenemase-producing Enterobacterales and P. aeruginosa in Spain, with practical implications for control and optimal treatment of infections caused by these MDR organisms.Continuous efforts should be maintained in the future in this area, with a focus on maintaining the clinical efficacy of recently introduced new therapeutic agents.

FIGURE 1
FIGURE 1 Geographical distribution of carbapenemase-producing microorganisms.Distribution of carbapenemases in Enterobacterales.Distribution of carbapenemases in Pseudomonas aeruginosa.The map shows a pie chart at each participating hospital with the distribution and total number of carbapenemases in Enterobacterales (A) and Pseudomonas aeruginosa (B).*Pie charts corresponding to the Community of Madrid participating hospitals are shown at the side of the map in order to avoid overlaping.

TABLE 1
Susceptibility methods used for testing the indicated number (n) and percentages (%) of evaluated isolates in the participating centres.

TABLE 2
Methods for carbapenemase identification.

TABLE 3
Microorganisms and type of carbapenemase isolated (n/%) in Enterobacterales and Pseudomonas aeruginosa.

TABLE 4
Clinical categories of antimicrobial agents reported by the participating centres by family of carbapenemase-producing microorganisms.